SBB12Ntbk-MelissaMilder

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Contents

~~!~~

Melissa Milder 22:16, 31 January 2012 (EST)

This is my next entry.

Melissa Milder 14:38, 16 February 2012 (EST)

1. Set up PCR reaction for sbb1232 ToxR-CI' part:
PCR ca998/osbb1232R on pBca9525-Bjc0005
Product is pcrpdt.

2. Set up PCA reaction for sbb1215 lz_AAAA-4 part:
PCA1 on o16, o17, o18
Product is pca1.

1.For Part sbb1232 ToxR-CI':

PCR ca998/osbb1232R on pBca9525-Bjc0005 (1586bp, pcrpdt)

Suspending the oligos
osbb1232R:

  • oligo arrived from IDT as as 28.5 nmol
  • added 285 uL ddH2O

--> Concentration of osbb1232R is now 100 uM

ca998:

  • already provided as 100 uM mixture


Following the Protocol: "Cloning by PCR"

Diluting the 100 uM oligos to 10uM

9uL ddH2O <br>
1uL 100uM oligo <br>


Setting up the following reaction in a PCR tube

24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL ca998, 10uM
1uL osbb1232R, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA (pBca9525-Bjc0005)

-->PCR tube was labeled "ToxR-CI'"(top)/ "pcrpdt"(side) and categorized as 1kb-2kb.

PCR completed overnight

2. For Part sbb1215 lz_AAAA-4:

PCA1 on o16, o17, o18 (pca1)

Suspending the oligos
o16:

  • oligo arrived from IDT as as 83.3 nmol
  • added 833 uL ddH2O

--> Concentration of o16 is now 100 uM

017:

  • already provided as 100 uM mixture


o18:

  • oligo arrived from IDT as as 95.1 nmol
  • added 951 uL ddH2O

--> Concentration of osbb1232R is now 100 uM

Following the Protocol: "PCA Gene Synthesis-Assembly"

Setting up the following in a PCR tube

38 uL ddH2O
5 ul 10x expand buffer
5 ul 2mM dNTPs
1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks)
0.75 ul Expand polymerase

-->PCR tube was labeled "lz_AAAA-4" (top)/ "pca1"(side)

PCA completed overnight

Melissa Milder 20:34, 17 February 2012 (EST)

1. Analytical Gel for sbb1232 ToxR-CI' part.
2. Small Part Zymo Clean-up of pca1 for sbb1215 lz_AAAA-4 part and set up pca2.

1. For Part sbb1232 ToxR-CI':

  • PCR was completed overnight.
  • pcrpdt was prepared and run on Analytical Gel.

Analytical Gel Preparation

  1. 6uL of pcrpdt and 4uL of blue dye were mixed together in a pcr tube.
  2. 10uL of mixture was pipetted into the 3rd well from the left on the gel.
  3. Gel was run for 10 minutes, but had to be stopped early due to time constraints.

-->Gel may have to be run again.

Expected Results

  • Should observe a band from the pcrpdt that is 1586bp in length (should not have moved very far on the gel) along with 2 bands that are 22bp and 32bp from the oligos ca998 and osbb1232R, respectively.

Image:Gel2_from_class_2_17_2012.jpg

  • My sample is shown in well #3. Because the gel was not run for very long, the 2 smaller bands appear as one band at the bottom, while the larger band appears as the top band. The smaller bands (the oligos) should have run a lot farther through the gel, but the gel was not run for long enough to observe this.

2. For Part sbb1215 lz_AAAA-4:

Following the Protocol: "Small Frag Zymo Cleanup"

  1. Add 100 uL of Zymo ADB buffer (brown bottle) to the pcr tube (labeled "pca1").
  2. Transfer into the Zymo column (small clear guys)
  3. Add 500uL of Ethanol and pipette up and down to mix
  4. spin through (15s), discard waste.
  5. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  6. spin through (15s), discard waste.
  7. Add 200 uL of Zymo Wash Buffer
  8. spin through, discard waste.
  9. spin for 90 seconds, full speed to dry.
  10. elute with water (spin 60s) into a fresh Eppendorf tube

-->amount of water for elution (amount in=amount out): ~50uL
-->eppendorf tube was labeled "lz_AAAA-4 pca1 zymo"

Following the Protocol: "PCA Gene Synthesis-Amplification"

Setting up the following in a PCR tube:

1 ul each outer oligo (o16 and o12)
1 ul purified pca product (pca1)
.5 ul phusion
10 ul 5x phusion buffer
5 ul 2mM dNTPs
32.5 ul H2O 

-->PCR tube was labeled "lz_AAAA-4" (top)/ "pca2"(side)

PCA completed overnight

Melissa Milder 14:45, 21 February 2012 (EST)

For lz_AAAA-4 part:

  1. Analytical gel of pca2
  2. Small Fragment Zymo Clean-up of pca2
  3. Digest pca2 with NheI and BamHI
  4. Freeze 1215dig in ADB Buffer
  5. Next class: start zymo clean-up

Parts from Construction File:
Digest pca2 (NheI/BamHI, L, 1215dig)

For ToxR-CI' part:

  1. Regular Zymo clean-up
  2. Next class: digest pcrpdt with NheI and BamHI, gel purify, zymo clean-up

1. For sbb1215 lz_AAAA-4 part:

1. pca2 was run on Analytical Gel

  • 6uL of pca2 and 4uL of blue dye were mixed together and pipetted into well #7 (from left)

Image:MM_Pca2_analytical_gel.jpg

  • Comparing the bands to those of the ladder (well #4), my band appears to be approximately 142bp long as it should be.
  • There should also be 2 bands shorter in length for the oligos, o16 and 012. These bands may have run off the gel.


2. Small Fragment Zymo Clean-up of pca2
Following the Protocol:

  1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction.
  2. Transfer into the Zymo column (small clear guys)
  3. Add 500uL of Ethanol and pipette up and down to mix
  4. spin through (15s), discard waste.
  5. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  6. spin through (15s), discard waste.
  7. Add 200 uL of Zymo Wash Buffer
  8. spin through, discard waste.
  9. spin for 90 seconds, full speed to dry.
  10. elute with water (spin 60s) into a fresh Eppendorf tube

-->amount of water for elution (amount in=amount out): 51uL (from pca reaction) - 6uL (used for analytical gel) = 45uL
-->eppendorf tube was labeled "lz_AAAA-4 pca2 zymo"

3. Digest pca2 with Nhe1 and BamH1
From construction file:
Digest pca2 (NheI/BamHI, L, 1215dig)

Following the Protocol "EcoRI/BamHI Digest of Wobble Products":
The following was set up in a PCR tube labeled "MM"(top)/"1215dig" (side):

  25uL eluted DNA
  5uL NEB Buffer 2
  1uL EcoRI
  1uL BamHI
  18uL H2O
  • Note: the amounts of reactants were adjusted because there was not enough eluted DNA to follow the given protocol.
  • The reaction was then mixed by slamming the tube upside down on the table and then spinning it down.
  • The reaction was then incubated at 37 degrees in the thermocycler for 1 hour.


4. Freeze 1215dig in ADB Buffer

  • Due to time constraints, the small fragment zymo clean-up could not be completed, so the reaction was mixed with 100uL of Zymo ADB buffer and then frozen.

2. For sbb1232 ToxR-CI' part:

1. Regular Zymo Clean-up of pcrpdt
Following the Protocol:

  1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
  2. Transfer into the Zymo column (small clear guys)
  3. spin through, discard waste.
  4. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  5. spin through, discard waste.
  6. Add 200 uL of Zymo Wash Buffer
  7. spin through, discard waste.
  8. spin for 90 seconds, full speed to dry.
  9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

-->amount of water for elution: (amount in=amount out): 33.6uL (from PCR reaction) - 6uL (used for analytical gel) = 27.6uL
-->eppendorf tube was labeled "ToxR-CI' pcrpdt zymo"

Melissa Milder 01:58, 24 February 2012 (EST)

For ToxR-CI' Part:

  1. Digest pcrpdt
  2. Gel purification of pcrdig
  3. Melt cut-out band with ADB buffer and freeze overnight
  4. Next time: continue with regular zymo

Parts from Construction File:
Digest pcrpdt (NheI/BamHI, 1083+492+11, M, pcrdig)
Digest pBca9525-Bca1834 (NheI/BamHI, L, vectdig)
Ligate pcrdig and vectdig, product is pBca9525-sbb1232

For lz_AAAA-4 Part:

  1. Small fragment zymo clean-up of 1215dig
  2. ligation of NheI/BamHI digests: 1215dig and vectdig
  3. Transformation of pBca9525-sbb1215

Parts from Construction File:
Digest pBca9525-Bca1834 (NheI/BamHI, L, vectdig)
Ligate 1215dig + vectdig, product is pBca9525-sbb1215

1. For Part sbb1232 ToxR-CI':

1. Digest pcrpdt
Following the protocol "EcoRI/BamHI Digest of PCR Products" (using NheI/BamHI instead):

  • Set up the following reaction in a medium sized eppendorf tube:
  8uL of eluted PCR product
  1uL of NEB Buffer 2
  0.5uL NheI
  0.5uL BamHI

-->The tube was labeled "MM" (top)/"ToxR-CI' pcrdig" (side)

  • Incubate at 37 degrees on the thermocycler for 1 hour

2. Gel purification of pcrdig

  • Set up an agarose gel and run it (my sample was run in well #1)

Image:2012_02_23_gel1_ssb2012spring.jpg

  • Note: My sample should have been run longer because the gel was not run long enough for the bands to separate as they should have into a small, medium, and large fragment. I should have cut out the medium fragment (492 bp); however, I cut out the entire region of DNA that was visualized on the gel, meaning my cut-out sample may contain all three of the bands. However, in this case, it is ok that my sample now contains all of the bands because only the medium sized band (492 bp) has sticky ends that are NheI and BamHI that are required to form a full circle with the vector digest (also containing sticky ends of NheI and BamHI) during ligation. The large and small bands will not interfere during the ligation because these fragments do not have matching ends to the vector digest fragment. The small and large DNA fragments will only have one matching sticky end to the vector digest and, thus, cannot form a complete circle of DNA during ligation. Thus, when the ligated DNA is transformed into cells, the cells will not form colonies unless they have complete circular DNA. Thus, only the DNA containing the 492 bp fragment will form colonies.

3. Melt cut-out band with ADB buffer and freeze overnight.

  • Melt cut-out band with 600uL ADB buffer at 55 degrees
  • Due to time constraints,the zymo clean-up process was stopped here and will be continued next class


2. For Part sbb1215 lz_AAAA-4:

1. Small Fragment Zymo Clean-up of 1215dig
Following the Protocol "Small-Frag Zymo Clean-up":

  1. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction. (already done last class)
  2. Transfer into the Zymo column (small clear guys)
  3. Add 500uL of Ethanol and pipette up and down to mix
  4. spin through (15s), discard waste.
  5. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  6. spin through (15s), discard waste.
  7. Add 200 uL of Zymo Wash Buffer
  8. spin through, discard waste.
  9. spin for 90 seconds, full speed to dry.
  10. elute with water (spin 60s) into a fresh Eppendorf tube

-->amount of water for elution (amount in=amount out): 25uL (from digestion reaction) -->eppendorf tube was labeled: "lz_AAAA-4 1215dig zymo"

2. Ligation of NheI/BamHI digests: 1215dig and vectdig

  • Ligation of 1215dig and pBca9525-Bca1834 vectdig (already digested for us)

Following the Protocol "Ligation of EcoRI/BamHI Digests" (used NheI/BamHI instead):

  • The reaction was set up in a medium sized eppendorf tube:
  6.5uL ddH2O
  1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
  1uL Vector digest (pBca9525-Bca1834 vectdig)
  1uL Insert digest (1215dig)
  0.5uL T4 DNA Ligase

-->eppendorf tube was labeled: "MM lz_AAAA-4 lig" (top)/"1215dig/vectdig ligation" (side)

  • Pound upside down on the bench to mix
  • Give it a quick spin to send it back to the bottom of the tube
  • Incubate on the benchtop for 30min
  • Put on ice and proceed to the transformation

3. Transformation of pBca9525-sbb1215
Following the Protocol "Transformation by Heat-Shock":

   1. Thaw a 100 uL aliquot of cells on ice
   2. Add 15 uL of KCM to the cells 
   3. Put your ligation mixture on ice, let cool a minute or two
   4. Add 50 uL of the cell cocktail to the ligation (tube labeled "MM lz_AAAA-4 lig"), stir to mix
   5. Let sit on ice for 10 min
   6. Heat shock for 90 seconds at 42 (longer incubation may work better)
   7. Put back on ice for 1 min
   8. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
   9. Plate 75 uL on selective antibiotics, let incubate at 37 degrees overnight

Melissa Milder 17:46, 24 February 2012 (EST)

For ToxR-CI' Part:

  1. Regular Part Zymo Clean-up of pcrdig
  2. Ready for ligation (next class)

For lz_AAAA-4 Part:

  1. Pick colonies
  2. Ready for DNA extraction (next class)

1. For Part sbb1232 ToxR-CI':

1. Regular Part Zymo Clean-up of ToxR-CI' pcrdig Following the Protocol "Regular Zymo Cleanup":

  1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction. (already done last time)
  2. Transfer into the Zymo column (small clear guys)
  3. spin through, discard waste.
  4. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
  5. spin through, discard waste.
  6. Add 200 uL of Zymo Wash Buffer
  7. spin through, discard waste.
  8. spin for 90 seconds, full speed to dry.
  9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

-->amount of water for elution (amount in=amount out): 8uL (from digestion of ToxR-CI' pcrpdt)
-->eppendorf tube was labeled "ToxR-CI' pcrdig zymo"

  • ready for ligation (next class)

2. For Part sbb1215 lz_AAAA-4:

1. Pick Colonies Expressing pBca9525-sbb1215 Plasmid
Following the Protocol "Picking of Colonies":

  • For each construct you will pick and later miniprep 2 colonies
  • Add 4mL of LB media with the appropriate antibiotics to a clean test tube
  • Pick a well-isolated, round, and "normal" looking colony with a toothpick
  • Drop it in the test tube
  • Incubate at 37 overnight

--> 4 colonies were picked and incubated

Melissa Milder 14:44, 28 February 2012 (EST)

For ToxR-CI' Part:

  1. Ligate ToxR-CI' pcrdig and NheI/BamHI vectdig
  2. Transformation of pBca9525-sbb1232 into cells by heat shock
  3. Plate cells on spectomycin antibiotic plate
  4. Incubate overnight
  5. Next time: check colonies and pick white colonies (if distinguishable from red colonies)

Parts from Construction File:
Ligate pcrdig and vectdig, product is pBca9525-sbb1232

For lz_AAAA-4 Part:

  1. Observe bacterial growth in liquid media
  2. Observe colonies from plate that were grown 2 lab days ago
  3. Mini-prep the white colonies that grew in tube #2 of liquid media
  4. Re-pick colonies for tubes #1, #3, and #4
  5. Next time: Mini-prep re-picked colonies and digest all mini-prepped products to check if the transformed vectors are correct


1. For Part sbb1232 ToxR-CI':

1. Ligate ToxR-CI' pcrdig and NheI/BamHI vectdig Following the protocol "Ligation of EcoRI/BamHI digests" (used NheI/BamHI instead):

  • The following reaction was set up in an eppendorf tube:
  6.5uL ddH2O
  1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
  1uL Vector digest
  1uL Insert digest
  0.5uL T4 DNA Ligase

-->the tube was labeled: "pBca9525-sbb1232 ligation"(top)/"ToxR-CI' ligation product"(side)

  • Pound upside down on the bench to mix
  • Give it a quick spin to send it back to the bottom of the tube
  • Incubate on the benchtop for 30min
  • Put on ice and proceed to the transformation


2. Transformation of pBca9525-sbb1232 into cells by heat shock Following the Protocol "Transformation by Heat Shock":

  1. Thaw a 100 uL aliquot of cells on ice
  2. Add 15 uL of KCM to the cells 
  3. Put your ligation mixture on ice, let cool a minute or two
  4. Add 70 uL of the cell cocktail to the ligation, stir to mix
  5. Mixture was transfered to a medium sized tube labeled "ToxR-CI' ligation"
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour


3. Plate cells on spectomycin antibiotic plate

  • Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight


4. Next time: check colonies and pick white colonies (if distinguishable from red colonies)

2. For Part sbb1215 lz_AAAA-4:

1. Observe bacterial growth in liquid media

  • All 4 tubes were cloudy/murky
  • Tubes #1, #3, and #4, all contained a red tint to them, while Tube #2 contained a yellow tint to it


2. Observe colonies from plate that were grown 2 lab days ago

  • When going back to the plate labeled "MM lz_AAAA-4 sbb1215 (spec)," it appears that some of the colonies are now distinguishably red compared to the white colonies (this was not noticeable before)
  • When observing the colonies that I had picked, it is clear that 3 of the picked colonies were now red and 1 of them was still white
  • From this, I concluded that the cultured tube #2 that had a yellow tint contained the white colony that I want.
  • Thus, I kept tube #2 for a mini-prep and disposed of the other 3 tubes. I also re-picked colonies for these 3 tubes.


3. Mini-prep the white colonies that grew in tube #2 of liquid media Following the Protocol "Miniprep Purification of DNA":

  1. Pellet 1.5 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube.
  2. Dump supernatant
  3. Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
  4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
  5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
  6. Spin in centrifuge at top speed for 5 minutes.
  • Note: After spinning, there were still white things floating around in the solution so I centrifuged it again for 5 minutes.
  1. Label blue columns with an alcohol-resistant lab pen. (labeled: "MM sbb1215 #2 Miniprep")
  2. Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
  3. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
  4. Wash each column with 500 uL of PB buffer.
  5. Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
  6. Wash with 750uL of PE buffer (washes the salts off the resins).
  7. Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
  8. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
  9. Label new Microcentrifuge tubes and put columns in them. (labeled: "MM sbb1215 #2 Miniprepped")
  10. Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
  11. Spin in centrifuge at top speed for 30 seconds.
  12. Take out columns and cap the tubes.
  13. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.


4. Re-pick colonies for tubes #1, #3, and #4 Following the Protocol "Picking of Colonies":

  • For each construct you will pick and later miniprep 4 colonies (note: I am now picking 3 because one was successful last time)
  • Add 4mL of LB media with the appropriate antibiotics to a clean test tube
  • Pick a well-isolated, round, and "normal" looking colony with a toothpick
  • Drop it in the test tube
  • Incubate at 37 overnight


5. Next time: Mini-prep re-picked colonies and digest all mini-prepped products to check if the transformed vectors are correct.

Melissa Milder 15:07, 1 March 2012 (EST)

For ToxR-CI' Part:

  1. Zack already picked 2 colonies.
  2. Miniprep both of these incubated bacteria samples in liquid media.
  3. Prepare for Analytical Digest
  4. Next Time: Analytical Digest


For lz_AAAA-4 Part:

  1. Observe 3 tubes of liquid media containing picked colonies for bacterial growth.
  2. Miniprep incubated bacteria samples in liquid media if bacterial growth was observed.
  3. Prepare for Analytical Digest
  4. Next Time: Analytical Digest


1. For Part sbb1232 ToxR-CI':

Zack already picked 2 colonies.

  • Didn't have to pick colonies and incubate in liquid media (already done)
  • Protocol for picking colonies is as follows ("Picking of Colonies"):
*For each construct you will pick and later miniprep 2 colonies
*Add 4mL of LB media with the appropriate antibiotics to a clean test tube
*Pick a well-isolated, round, and "normal" looking colony with a toothpick
*Drop it in the test tube
*Incubate at 37 overnight

-->tubes were labeled 1232-1 and 1232-2

  • both tubes showed bacterial growth! (liquid media was cloudy and yellow in appearance)


Miniprep both of these incubated bacteria samples in liquid media.
Following the Protocol "Miniprep Purification of DNA":

  1. Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube (labeled "MM 1232-1" and "MM 1232-2")
  2. Dump supernatant and repeat steps 1 and 2 for the remaining of the liquid media and cultured bacteria.
  3. Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly.
  4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
  5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
  6. Spin in centrifuge at top speed for 5 minutes.
  7. Label blue columns with an alcohol-resistant lab pen. (labeled "MM 1232-1 Miniprep" and "MM 1232-2 Miniprep")
  8. Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
  9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
  10. Wash each column with 500 uL of PB buffer.
  11. Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
  12. Wash with 750uL of PE buffer (washes the salts off the resins).
  13. Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
  14. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
  15. Label new Microcentrifuge tubes and put columns in them. (labeled "MM 1232-1"(top)/"Miniprepped" (side) and "MM 1232-2" (top)/"Miniprepped"(side))
  16. Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
  17. Spin in centrifuge at top speed for 30 seconds.
  18. Take out columns and cap the tubes.
  19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.


Prepare for Analytical Digest

  • Need to alter protocol for my specific part.
  • Amount of miniprepped plasmid necessary is 2uL instead of 4uL because there is high enough concentration of the plasmid present.
  • Determining what enzymes to use: Using NheI/BamHI for digestion gives a parent digest of 3494 bp and 841 bp and a product digest of 3494 bp and 492 bp. This is good enough to distinguish the two plasmids and the fragments are large enough that they will be visible and will not run off of the gel.


Next Time: Analytical Digest

2. For Part sbb1215 lz_AAAA-4:

Observe 3 tubes of liquid media containing picked colonies for bacterial growth.

  • All 3 tubes of liquid media and bacteria appeared clear and yellow after incubation. Thus, there was no bacterial growth! These samples could not be used.
  • I decided to just use my one successful sample that was miniprepped last class (from the first round of picking colonies for this sample). I may need to re-do the transformation if I was not successful in creating my part in this particular sample.

Miniprep incubated bacteria samples in liquid media if bacterial growth was observed.

  • no bacterial growth was observed so no additional minipreps were performed.


Prepare for Analytical Digest

  • Need to alter protocol for my specific part.
  • Amount of miniprepped plasmid necessary is 2uL instead of 4uL because there is high enough concentration of the plasmid present since lz_AAAA-4 is a pUC plasmid.
  • Determining what enzymes to use: Using EcoRI/BamHI for digestion gives a parent digest of 2472 bp and 1863 bp and a product digest of 2472 bp and 1148 bp. This is good enough to distinguish the two plasmids and the fragments are large enough that they will be visible and will not run off of the gel.


Next Time: Analytical Digest

  • Note: When looking back at the construction file, I noticed a typo that may have caused my results to be completely different from what they are expected to be.

Construction file:

PCA1 on o16,o17,o18       (pca1)
PCA2 with o16/o12 on pca1 (142 bp, pca2)
Digest pca2               (NheI/BamHI, L, 1215dig)
Digest pBca9525-Bca1834   (NheI/BamHI, L, vectdig)
Ligate 1215dig + vectdig, product is pBca9525-sbb1215
  • The PCA2 step should have been done with o16/o18 on pca1 instead of o16/o12 on pca1. Thus, the resulting product from this procedure will not be the expected part; however, the size of the bands after digestion will still be the same.


Melissa Milder 15:33, 2 March 2012 (EST)

For ToxR-CI' Part:

  1. Analytical Digest of 1232-1 and 1232-2.
  2. Run Analytical Gel (Mapping)


For lz_AAAA-4 Part:

  1. Analytical Digest of 1215-2.
  2. Run Analytical Gel (Mapping)


1. For Part sbb1232 ToxR-CI':

Analytical Digest of 1232-1 and 1232-2

  • Analytical Digests were set up for both parts (1232-1 and 1232-2)

Following the Protocol "Analytical Digests (Mapping)" (with adjustments for my particular sample): Set up the following 10uL reaction in a PCR tube:

6uL ddH2O
2uL Miniprepped plasmid (1232-1 and 1232-2 separately)
1uL 10x NEB Buffer 2
.5uL NheI
.5uL BamHI

-->PCR tubes were labeled "1232-1 MM"(top)/"anal dig"(side) and "1232-2 MM"(top)/"anal dig"(side) for both of the samples, respectively

  • Incubate at 37 on the thermocycler for 30 minutes


Run Analytical Gel (Mapping)

  • Set-up an analytical gel for these 2 parts (mixing 6uL of sample and 4uL of blue dye and pipetting the mixture into separate wells on the analytical gel)
  • Sample 1232-1 digest is in well#3, while sample 1232-2 digest is in well#4
  • Take a picture of the gel

Image:2012_03_02_gel2_ssb2012spring.jpg

  • Calculate the expected fragment sizes: The expected fragment sizes are 3494 bp and 492 bp. Make sure it doesn't look like the parent digest, which would have fragment sizes of 3494 bp and 841 bp.
  • Are the calculated sizes consistent with the bands on the gel?
  • After observing the bands on this gel, it appears that the fragment sizes may be close to 3494 bp and 841 bp, suggesting that the DNA fragments expressed may just be from the parent digest.


2. For Part sbb1215 lz_AAAA-4:

Analytical Digest of 1215-2
Following the Protocol "Analytical Digests (Mapping)" (with adjustments for my particular sample): Set up the following 10uL reaction in a PCR tube:

6uL ddH2O
2uL Miniprepped plasmid (1232-1 and 1232-2 separately)
1uL 10x NEB Buffer 2
.5uL EcoRI
.5uL BamHI

-->PCR tube was labeled "1215-2 MM"(top)/"anal dig"(side)

  • Incubate at 37 on the thermocycler for 30 minutes


Run Analytical Gel (Mapping)

  • Set-up an analytical gel for this sample (mixing 6uL of sample and 4uL of blue dye and pipetting the mixture into separate wells on the analytical gel)
  • Sample 1215-2 digest is in well#5
  • Take a picture of the gel

Image:2012_03_02_gel2_ssb2012spring.jpg

  • Calculate the expected fragment sizes: The expected fragment sizes are 2472 bp and 1148 bp. Make sure it doesn't look like the parent digest, which would have fragment sizes of 2472 bp and 1863 bp.
  • Are the calculated sizes consistent with the bands on the gel?
  • The gel shows that there were three bands present on the gel, all of lengths greater than 1.0 kb. This shows that the result of this was not as expected.


Melissa Milder 13:42, 6 March 2012 (EST)

1. Observed Analytical Gels Again
2. Digested Miniprepped Products for Both Parts Again
3. Ran Another Analytical Gel for Both Digested Parts

1. For Part sbb1232 ToxR-CI':

Observed Analytical Gels Again

  • See last notebook entry for gel pictures
  • The two samples of sbb1232 are shown in wells 3 and 4
  • It appears that the small band is between 500bp and 1kb and not the expected 492 bp; however, the ladder appears to be overloaded and difficult to read. It is not a good comparison. It is possible that the smaller band of the two shown is 492 bp. To be sure of this, I ran another Analytical Digest and Gel.


Analytical Digest Miniprepped Products for Both Parts 1232-1 and 1232-2 Again

  • The protocol "Analytical Digests (Mapping)" was altered so that less of the DNA was used for the digest because the last analytical digest showed a high concentration of the DNA. The protocol was altered as follows:

Set up the following 10uL reaction in a PCR tube:

7uL ddH2O
1uL Miniprepped plasmid (1232-1 and 1232-2 separately)
1uL 10x NEB Buffer 2
.5uL NheI
.5uL BamHI

-->PCR tubes were labeled "1232-1 MM"(top)/"anal dig"(side) and "1232-2 MM"(top)/"anal dig"(side) for both of the samples, respectively

  • Incubate at 37 on the thermocycler for 30 minutes


Ran Another Analytical Gel for Both Digested Parts

  • Well #1 is 1232-1 and well #2 is 1232-2
  • Set-up an analytical gel for these 2 parts (mixing 6uL of sample and 4uL of blue dye and pipetting the mixture into separate wells on the analytical gel)
  • This time, used 2uL of the ladder in order to make the ladder appear more clear and prevent overloading of the ladder
  • Take a picture of the gel

Image:NEB_2-log_ladder.gifImage:2012_03_06_gel4_ssb2012spring.jpg

  • Calculate the expected fragment sizes: The expected fragment sizes are 3494 bp and 492 bp. Make sure it doesn't look like the parent digest, which would have fragment sizes of 3494 bp and 841 bp.
  • Are the calculated sizes consistent with the bands on the gel?
  • By observing the analytical gel, it appears that the two bands observed are ~3500 bp and ~500 bp as expected.
  • Next time: one of these two samples should be prepared for sequencing.


2. For Part sbb1215 lz_AAAA-4:

Observed Analytical Gels Again

  • See last notebook entry for gel pictures
  • The sample of sbb1215-2 is shown in well 5
  • It appears that three bands are shown, two of which appear to be above 3 kb and one that appears to be around 2 kb. I was expecting to see only two bands that are 2472 bp and 1148 bp and possibly another band that was 3620 bp if some of the plasmids were only digested by one enzyme. This may have been due to the ladder being inaccurate because it may have been overloaded. In order to check this, I ran my digest again using less of the miniprepped DNA product and less of the ladder for the analytical gel so that the ladder did not come out overloaded


Analytical Digest Miniprepped Products for Part 1215-2 Again

  • The protocol "Analytical Digests (Mapping)" was altered so that less of the DNA was used for the digest because the last analytical digest showed a high concentration of the DNA. The protocol was altered as follows:

Set up the following 10uL reaction in a PCR tube:

7uL ddH2O
1uL Miniprepped plasmid (1215-2)
1uL 10x NEB Buffer 2
.5uL EcoRI
.5uL BamHI

-->PCR tube was labeled "1215-2 MM"(top)/"anal dig"(side)

  • Incubate at 37 on the thermocycler for 30 minutes


Ran Another Analytical Gel for Digested Parts

  • Well #3 is 1215-2
  • Set-up an analytical gel (mixing 6uL of sample and 4uL of blue dye and pipetting the mixture into separate wells on the analytical gel)
  • This time, used 2uL of the ladder in order to make the ladder appear more clear and prevent overloading of the ladder
  • Take a picture of the gel

Image:NEB_2-log_ladder.gifImage:2012_03_06_gel4_ssb2012spring.jpg

  • Calculate the expected fragment sizes: The expected fragment sizes are 2472 bp and 1148 bp. Make sure it doesn't look like the parent digest, which would have fragment sizes of 2472 bp and 1863 bp.
  • Are the calculated sizes consistent with the bands on the gel?
  • By observing the gel, it seems like there are five bands present when we only expected there to be 2 or 3 bands present. Four of these bands appear to be between 3.0 kb and 10 kb, but none of the expected bands should be this large, except for a fragment that may have only been digested by one enzyme. The fifth band appeared to be around 1.2 kb.
  • Next time: For this part, it may be necessary to pick colonies again and extract the DNA again.


Melissa Milder 13:20, 8 March 2012 (EST)

For ToxR-CI' Part:

  1. Prepare Miniprepped 1232-1 and 1232-2 for Sequencing


For lz_AAAA-4 Part:

  1. Observe Bands from last Analytical Gel
  2. Run Another Analytical Digest of 1215-2.
  3. Run Analytical Gel (Mapping)


1. For Part sbb1232 ToxR-CI':

Prepare Miniprepped 1232-1 and 1232-2 for Sequencing

  • pipette miniprepped product plasmids into plates for sequencing (Row 4, Column 1) on separate plates.
  • enter parts into Clotho as pBca9525-sbb1232 for designated for row 4, column 1 of the two tables.


2. For Part sbb1215 lz_AAAA-4:

Observe Bands from last Analytical Gel

  • It appears that there were five bands present, two of which appear to be 2.5kb and 1.2kb as expected from the digested product plasmid.
  • The other bands present may have been due to another plasmid present in the cells or other arrangements of the digested fragments.
  • In order to check for this, I tried digesting the miniprepped product with different enzymes. This time, I used NhEI and EcoRI. These enzymes should produce a digested product with fragments of 2598 bp and 1022 bp.

Run Another Analytical Digest of 1215-2

  • The protocol "Analytical Digests (Mapping)" was altered from last time by using NheI and EcoRI as the enzymes. The protocol was altered as follows:

Set up the following 10uL reaction in a PCR tube:

7uL ddH2O
1uL Miniprepped plasmid (1215-2)
1uL 10x NEB Buffer 2
.5uL EcoRI
.5uL NheI

-->PCR tube was labeled "1215-2 MM"(top)/"anal dig"(side)

  • Incubate at 37 on the thermocycler for 30 minutes


Run Analytical Gel (Mapping)

  • Well #6 is MM 1215-2
  • Set-up an analytical gel (mixing 6uL of sample and 4uL of blue dye and pipetting the mixture into separate wells on the analytical gel)
  • This time, used 2uL of the ladder in order to make the ladder appear more clear and prevent overloading of the ladder
  • Take a picture of the gel

Image:NEB_2-log_ladder.gifImage:2012_03_08_gel2_ssb2012spring.jpg

  • Calculate the expected fragment sizes: The expected fragment sizes are 2598 bp and 1022 bp.
  • Are the calculated sizes consistent with the bands on the gel?
  • Because there are so many bands present, it is likely that there were digestion problems that occurred, producing unexpected bands. These unexpected bands are most likely the larger bands. When looking at the smaller bands and comparing their sizes to the sizes on the ladder, the sizes still seem too large to be 2598 bp and 1022 bp; however, we think that there have been problems going on with the ladder. Thus, I compared my bands to the expected sizes of the other peoples' bands that were run on this gel. Au dig SOE3 should be 1.4 kb, JK should be 1.4 kb and 2.5 kb, JS 1201 should be 1092 bp and 627 bp and JS 1226 should be 1408 bp and 1640 bp. Thus, it is likely that my two smallest bands shown on the gel could be ~2.5 kb and ~1.0 kb as expected. The largest band is most likely genomic DNA.
  • Because these extra bands are present (likely due to a bad miniprep or a bad digestion), I need to retransform the DNA.
  • Next time: retransform DNA and pick colonies from old plate.


Melissa Milder 13:18, 13 March 2012 (EDT)

For ToxR-CI' Part:

  1. Waiting for sequencing results

For lz_AAAA-4 Part:

  1. Re-do ligation of NheI/BamHI digests: 1215dig and vectdig
  2. Retransformation of pBca9525-sbb1215

Also try:

  1. Re-pick colonies from plate

1. For Part sbb1215 lz_AAAA-4:

Ligation of NheI/BamHI digests: 1215dig and vectdig

  • Ligation of 1215dig and pBca9525-Bca1834 vectdig (already digested for us)

Following the Protocol "Ligation of EcoRI/BamHI Digests" (used NheI/BamHI instead):

  • The reaction was set up in a medium sized eppendorf tube:
  6.5uL ddH2O
  1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
  1uL Vector digest (pBca9525-Bca1834 vectdig)
  1uL Insert digest (1215dig)
  0.5uL T4 DNA Ligase

-->eppendorf tube was labeled: "MM lz_AAAA-4 lig" (top)/"1215dig/vectdig ligation" (side)

  • Pound upside down on the bench to mix
  • Give it a quick spin to send it back to the bottom of the tube
  • Incubate on the benchtop for 30min
  • Put on ice and proceed to the transformation


Transformation of pBca9525-sbb1215
Following the Protocol "Transformation by Heat-Shock":

   1. Thaw a 100 uL aliquot of cells on ice
   2. Add 15 uL of KCM to the cells 
   3. Put your ligation mixture on ice, let cool a minute or two
   4. Add 50 uL of the cell cocktail to the ligation (tube labeled "MM lz_AAAA-4 lig"), stir to mix
   5. Let sit on ice for 10 min
   6. Heat shock for 90 seconds at 42 (longer incubation may work better)
   7. Put back on ice for 1 min
   8. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
   9. Plate 75 uL on selective antibiotics, let incubate at 37 degrees overnight

-->set up incubation tubes for Zack to pick: labeled MM 1215-1**, MM 1215-2**, MM 1215-3**, MM 1215-4**

Re-picking Colonies Expressing pBca9525-sbb1215 Plasmid
Following the Protocol "Picking of Colonies":

  • For each construct you will pick and later miniprep 2 colonies
  • Add 4mL of LB media with the appropriate antibiotics to a clean test tube
  • Pick a well-isolated, round, and "normal" looking colony with a toothpick
  • Drop it in the test tube
  • Incubate at 37 overnight

--> 4 colonies were picked and incubated: labeled MM 1215-1*, MM 1215-2*, MM 1215-3*, MM 1215-4*
Next time: Miniprep and Analytical Digest

Melissa Milder 13:51, 15 March 2012 (EDT)

For ToxR-CI' Part:

  1. Waiting for sequencing results

For lz_AAAA-4 Part:

  1. Observe bacterial growth from 1215-#* test tubes (repicked from original plate) and from 1215-#** test tubes (picked from new plate)
  2. Miniprep samples in which bacterial growth was observed
  3. Prepare for analytical digest
  4. Next time: analytical digest


1. For Part sbb1215 lz_AAAA-4:

Observe Bacterial Growth

  • no bacterial growth was observed in the tubes labeled 1215-#* (from the bacteria that were re-picked from the original plate)
  • bacterial growth was observed in the tubes labeled 1215-#** (from the bacteria that were picked from the new plate)


Miniprep Samples with Bacterial Growth

  • The four tubes labeled 1215-1**, 1215-2**, 1215-3**, and 1215-4** were all miniprepped as defined in the protocol "Miniprep Purification of DNA:
  1. Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube (labeled "MM 1215-1**", "MM 1215-2**", "MM 1215-3**", and "MM 1215-4**")
  2. Dump supernatant and repeat steps 1 and 2 for the remaining of the liquid media and cultured bacteria.
  3. Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly.
  4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
  5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
  6. Spin in centrifuge at top speed for 5 minutes.
  7. Label blue columns with an alcohol-resistant lab pen. (labeled "MM 1215-1** Miniprep", "MM 1215-2** Miniprep", "MM 1215-3** Miniprep", and "MM 1215-4** Miniprep")
  8. Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
  9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
  10. Wash each column with 500 uL of PB buffer.
  11. Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
  12. Wash with 750uL of PE buffer (washes the salts off the resins).
  13. Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
  14. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
  15. Label new Microcentrifuge tubes and put columns in them. (labeled "MM 1215-1**"(top)/"Miniprepped" (side), "MM 1215-2**"(top)/"Miniprepped" (side), "MM 1215-3**"(top)/"Miniprepped" (side), and "MM 1215-4**"(top)/"Miniprepped" (side))
  16. Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
  17. Spin in centrifuge at top speed for 30 seconds.
  18. Take out columns and cap the tubes.
  19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.


Prepare Samples for Analytical Digest

  • Same as before:

Determining what enzymes to use: Using EcoRI/BamHI for digestion gives a parent digest of 2472 bp and 1863 bp and a product digest of 2472 bp and 1148 bp. This is good enough to distinguish the two plasmids and the fragments are large enough that they will be visible and will not run off of the gel. Can also try using EcoRI/NheI for digestion, which gives a product digest of 2598 bp and 1022 bp.
Next time: Analytical Digest

Melissa Milder 12:41, 20 March 2012 (EDT)

For ToxR-CI' Part:

  1. Work on sequencing data


For lz_AAAA-4 Part:

  1. Analytical Digest of 1215-1**, 1215-2**, 1215-3**, and 1215-4**.
  2. Run Analytical Gel (Mapping)


1. For Part sbb1232 ToxR-CI':

  • Data was sequenced: data matched expected sequence perfectly! Perfect part!

2. For Part sbb1215 lz_AAAA-4:

Run Analytical Digest of 1215 Minipreps

  • The protocol "Analytical Digests (Mapping)" was performed using EcoRI/NheI as enzymes. The protocol is as follows:

Set up the following 10uL reaction in a PCR tube:

7uL ddH2O
1uL Miniprepped plasmid (1215-2)
1uL 10x NEB Buffer 2
.5uL EcoRI
.5uL NheI

-->PCR tubes were labeled "MM 1"(top)/"1215-1** anal dig"(side), "MM 2"(top)/"1215-2** anal dig"(side), "MM 3"(top)/"1215-3** anal dig"(side), and "MM 4"(top)/"1215-4** anal dig"(side)

  • Incubate at 37 on the thermocycler for 30 minutes


Run Analytical Gel (Mapping)

  • Well #2 is MM 1215-1**, well #3 is MM 1215-2**, well #4 is MM 1215-3**, well #5 is MM 1215-4**
  • Set-up an analytical gel (mixing 6uL of sample and 4uL of blue dye and pipetting the mixture into separate wells on the analytical gel)
  • Add 10uL of the diluted ladder to well #6
  • Take a picture of the gel

Image:NEB_2-log_ladder.gifImage:2012_03_20_gel1_ssb2012spring.jpg

  • Calculate the expected fragment sizes: The expected fragment sizes are 2598 bp and 1022 bp.
  • Are the calculated sizes consistent with the bands on the gel?
  • Yes, the bands all appear to be about 2598 bp and 1022 bp, assuming that there was a small skew in the movement of the bands due to unbalanced electrodes (my four samples seem to be digested in the same way, but they were exposed to the electrode potentials at different amounts).


Melissa Milder 12:42, 22 March 2012 (EDT)

Sequencing!

Melissa Milder 14:33, 3 April 2012 (EDT)

Team 1:Quantify dimerization transduction for all ToxR-peptide constructs.

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